Assay for glycosylation deficiency disorders

Abstract

A method is provided for detecting carbohydrate-deficient glycoproteins in samples taken from subjects with metabolic disorders, such as alcohol abuse and subjects who display a syndrome of carrying abnormal levels of carbohydrate deficient glycoproteins. The method involves steps of reglycosylating with a fluorescent-conjugate deglycosylated glycoproteins in a sample of body fluid from a subject. A further step involves fluorometric detection of fluoresceinylated carbohydrates incorporated into truncated serum glycoproteins.

What is claimed is: 
     
       1. A method for detecting carbohydrate-deficient glycoproteins in a subject with a metabolic disorder, said method comprising the steps of: (a) obtaining a sample of body fluid from a subject, said sample comprising deglycosylated glycoprotein;   (b) reglycosylating with a fluorescent-labeled conjugate a portion of said deglycosylated glycoprotein;   (c) detecting the reglycosylated glycoprotein made in step (b).   
     
     
       2. The method of claim 1 wherein said step of detecting comprises quantitating the amount of reglycosylated glycoprotein. 
     
     
       3. The method of claim 1 wherein said sample of body fluid is serum. 
     
     
       4. The method of claim 1 wherein said reglycosylating comprises contacting said sample with a mixture comprising a glycosylation enzyme and said fluorescent-labeled conjugate under conditions sufficient to incorporate the fluorescent label into a portion of said deglycosylated glycoprotein, wherein said fluorescent-labeled conjugate is a nucleotide carbohydrate-fluorescent conjugate. 
     
     
       5. The method of claim 4 wherein said glycosylation enzyme is a glycosyltransferase. 
     
     
       6. The method of claim 4 wherein said nucleotide carbohydrate-fluorescent conjugate is a fluorescent CMP-glycoside. 
     
     
       7. The method of claim 4 wherein said nucleotide carbohydrate-fluorescent conjugate is CMP-9-Fluoresceinyl-AcNeu. 
     
     
       8. The method of claim 1 wherein said reglycosylating comprises the steps of (a) contacting said sample with a first mixture comprising a first glycosylation enzyme and a first nucleotide-sugar conjugate to produce a first reglycosylated glycoprotein; and   (b) contacting the first reglycosylated glycoprotein with a second mixture comprising a second glycosylation enzyme and a nucleotide carbohydrate-fluorescent conjugate under conditions sufficient to incorporate fluoresceinylated carbohydrate into a portion of said first reglycosylated glycoprotein.   
     
     
       9. The method of claim 8 wherein said first nucleotide-sugar conjugate is an unlabelled UDP-glycoside. 
     
     
       10. The method of claim 9 wherein said first nucleotide-sugar conjugate is UDP-galactose. 
     
     
       11. The method of claim 8 wherein said first and second glycosylation enzymes are glycosyltransferases. 
     
     
       12. The method of claim 11 wherein said first glycosylation enzyme is galactosyltransferase. 
     
     
       13. The method of claim 11 wherein said second glycosylation enzyme is sialyltransferase. 
     
     
       14. The method of claim 8 wherein said nucleotide carbohydrate-fluorescent conjugate is a fluorescent CMP-glycoside. 
     
     
       15. The method of claim 8 wherein said nucleotide carbohydrate-fluorescent conjugate is CMP-9-Fluoresceinyl-AcNeu. 
     
     
       16. A method for diagnosing a metabolic disorder in a subject, said method comprising the steps of: (a) obtaining a sample of body fluid from a subject, said sample comprising deglycosylated glycoprotein;   (b) reglycosylating with a fluorescent-labeled conjugate a portion of said deglycosylated glycoprotein to produce a reglycosylated glycoprotein;   (c) quantitating the reglycosylated glycoprotein made in step (b);   (d) comparing the amount of reglycosylated glycoprotein quantitated in step (c) to the amount of reglycosylated glycoprotein in samples of body fluid from normal subjects and subjects known to have said metabolic disorder.   
     
     
       17. The method of claim 16 wherein said sample of body fluid is serum. 
     
     
       18. The method of claim 16 wherein said reglycosylating comprises contacting said sample with a mixture comprising a glycosylation enzyme and said fluorescent-labeled conjugate under conditions sufficient to incorporate the fluorescent label into a portion of said deglycosylated glycoprotein, wherein said fluorescent-labeled conjugate is a nucleotide carbohydrate-fluorescent conjugate. 
     
     
       19. The method of claim 18 wherein said glycosylation enzyme is a glycosyltransferase. 
     
     
       20. The method of claim 19 wherein said glycosylation enzyme is sialyltransferase. 
     
     
       21. The method of claim 18 wherein said nucleotide carbohydrate-fluorescent conjugate is a fluorescent CMP-glycoside. 
     
     
       22. The method of claim 18 wherein said nucleotide carbohydrate-fluorescent conjugate is CMP-9-Fluoresceinyl-AcNeu. 
     
     
       23. The method of claim 16 wherein said reglycosylating comprises the steps of (a) contacting said sample with a first mixture comprising a first glycosylation enzyme and a first nucleotide-sugar conjugate to produce a first reglycosylated glycoprotein; and   (b) contacting the first reglycosylated glycoprotein with a second mixture comprising a second glycosylation enzyme and a nucleotide carbohydrate-fluorescent conjugate under conditions sufficient to incorporate fluoresceinylated carbohydrate into a portion of said first reglycosylated glycoprotein.   
     
     
       24. The method of claim 23 wherein said first and second glycosylation enzymes are glycosyltransferases. 
     
     
       25. The method of claim 24 wherein said first glycosylation enzyme is galactosyltransferase. 
     
     
       26. The method of claim 24 wherein said second glycosylation enzyme is sialyltransferase. 
     
     
       27. The method of claim 23 wherein said first nucleotide-sugar conjugate is an unlabelled UDP-glycoside. 
     
     
       28. The method of claim 27 wherein said first nucleotide-sugar conjugate is UDP-galactose. 
     
     
       29. The method of claim 23 wherein said nucleotide carbohydrate-fluorescent conjugate is a fluorescent CMP-glycoside. 
     
     
       30. The method of claim 23 wherein said nucleotide carbohydrate-fluorescent conjugate is CMP-9-Fluoresceinyl-AcNeu. 
     
     
       31. The method of claim 16 wherein the step of quantitating comprises measuring fluorescence emission from said reglycosylated glycoprotein. 
     
     
       32. The method of claim 16 wherein said metabolic disorder is alcoholism. 
     
     
       33. The method of claim 16 wherein said deglycosylated glycoprotein is transferrin. 
     
     
       34. The method of claim 16 wherein said metabolic disorder is a carbohydrate-deficient glycoprotein syndrome.